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Cell, Tumor, and Stem Cell Biology |
1 Biotechnology and 2 Radiation Protection Unit, Ente per le Nuove Tecnologie, l'Energia e l'Ambiente, CR-Casaccia, Rome, Italy and 3 Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy
Requests for reprints: Anna Saran, Biotechnology Unit, Ente per le Nuove Tecnologie, l'Energia e l'Ambiente, CR-Casaccia, Via Anguillarese 301, 00060 Rome, Italy. Phone: 39-06-3048-4304; Fax: 39-06-3048-3644; E-mail: saran{at}casaccia.enea.it.
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Introduction |
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The Patched (Ptc1) protein is a Sonic Hedgehog (Shh) receptor and negative regulator of Shh signaling. Ptc1-negative regulation is relieved on Shh binding, or after Ptc1 mutational inactivation (3). In the skin, the Shh pathway regulates hair follicle morphogenesis and growth, and Shh-inappropriate activation results in formation of BCC, classically considered a hair follicle–derived tumor (4). In vertebrates, transcriptional responses to Shh and other hedgehog proteins are mediated by downstream transcription factors of the Gli family (5). Gli1 and Gli2 act as transcriptional activators whereas Gli3 acts as a repressor. Overexpression of Gli1 or Gli2 in skin results in BCC-like tumor development (5, 6).
Mouse models of skin cancer in many instances are good approximations of human cancer counterparts and recapitulate the tumor-stromal interactions, angiogenesis, and the multistep nature of progression of many tumors (7). In particular, mice heterozygous for mutations in the Ptc1 gene (8, 9) represent an important model for BCC, as they exhibit an epidermal proliferative disorder, and develop BCC-like tumors following UV (9) or ionizing radiation (10). Thus, they facilitate the identification of genetic and cellular events of BCC tumorigenesis.
Mammalian skin is maintained through adult life by stem cells that have the capacity of self-renewal and also generate one or more cell lineages of the tissue (e.g., cells giving rise to the epidermis, the hair follicles, and the sebaceous glands; refs. 11–13). A specialized region of the hair follicle, known as the bulge, is a major repository of clusters of stem cells, whereas in the interfollicular epidermis, stem cells are thought to reside as single cells (13–17). Whereas the epidermis grows continuously, hair follicles are characterized by cyclic growth that alternates periods of growth (anagen), regression (catagen), and rest (telogen) throughout life (18). Although different stem cell populations have been defined in the skin, bulge stem cells are considered the primary target in cutaneous carcinogenesis (19). Because proliferating cells are more susceptible to tumor initiation (20–22), the transient activation of follicular stem cells to proliferate at the beginning of anagen suggests that the skin may be more susceptible to BCC induction by radiation during this time period.
The synchronous hair follicle cycling of the young mouse (18) provides opportunity to study how epidermal stem cell proliferation and functionality influence BCC development. Therefore, we have exposed groups of Ptc1neo67/+ mice to X-rays at different ages (3, 35, or 60 days), corresponding to periods of growth or quiescence of the follicles during the first and second hair cycles. Mice in the telogen phase of hair growth (60 days old) were also exposed at day 9 after induction of anagen by depilation. We find that not only the frequency of BCC is influenced by hair cycle phase at irradiation but also that different histologic BCC subtypes occur by irradiating the skin in particular hair cycle phases. Moreover, by examining the histochemical profile of normal skin and BCC histologic variants, we provide new insights into the relationship between target cells and BCC phenotype, strongly reinforcing the concept that the diversity of epidermal tumors reflects the range of differentiated lineages of the stem cells progeny.
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Materials and Methods |
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Irradiation. X-ray irradiation (half-value layer, 1.6 mmCu) was done using a Gilardoni CHF 320G X-ray generator (Gilardoni S.p.A., Mandello del Lario, Lecco, Italy) operated at 250 kVp, 15 mA, with filters of 2.0 mm Al and 0.5 mm Cu. Ptc1neo67/+ mice of both sexes were whole-body irradiated with 3 Gy of X rays at the estimated time of the second anagen [i.e., 35 days (n = 60)] or of the second telogen [i.e., 60 days (n = 56)]. An additional group (n = 54) was irradiated at 3 days (i.e., during the first anagen); to minimize mortality for medulloblastoma, a brain tumor highly inducible by radiation in neonatal mice (23), 3-mm-thick lead shields were accurately positioned to provide protection of mouse head during irradiation. Finally, Ptc1neo67/+ mice (n = 54) were irradiated at day 9 after induction of anagen in telogen skin by depilation. Briefly, mice in the telogen phase of hair growth (60 days old) were anesthetized and depilated on dorsal skin by hot depilatory wax (18). Twenty-seven heterozygotes were left untreated as controls.
Histologic analysis and tumor quantification. Mice were observed daily for their whole life span; at the first signs of morbidity, they were sacrificed and complete autopsies were done. Grossly normal appearing dorsal skin (5 cm2) and any skin or other tissue tumors were fixed in 4% buffered formalin, embedded in paraffin, sectioned, and stained with H&E. Tumor variables were expressed as the percentage of mice bearing one or more macroscopic or microscopic BCC-like tumors (incidence) and the mean number of macroscopic tumors per positive mouse (multiplicity). The incidence and multiplicity of microscopic BCCs were determined by analyzing dorsal skin samples from Ptc1neo67/+ mice (n = 131) autopsied in the time interval of macroscopic BCC appearance (15-87 weeks postirradiation). To shed light on hair cycle effects on nodular BCC appearance, we evaluated the same variables on the subset of mice from the three groups that were autopsied from 15 to 30 weeks postirradiation (n = 61). Briefly, three skin sections per mouse were analyzed; to ensure representative sampling, sections were recovered with intervals of 100 µm. Eighteen digital images per mouse, corresponding to 40 mm of transverse skin, were collected by IAS image processing software (Delta Sistemi, Rome, Italy) with a Nikon microscope (Eclypse E800, 4x objective). The number of independent tumors in the skin analyzed was averaged for calculation of tumor multiplicity (±SE).
Immunohistochemistry. Immunohistochemical analysis of tumors and normal skin was done as described (10). Antibodies included rabbit polyclonal anti–cytokeratin 14 (CK14; 1:500 dilution; BabCo, Berkeley, CA), monoclonal anti-CK15 (1:100; Abcam Ltd., Cambridge, United Kingdom), and anti–
-smooth muscle actin (SMA; 1:400; Sigma Chemical Co., St. Louis, MO).
Loss of heterozygosity at the Ptc1 locus. Samples from all macroscopic BCC-like tumors and noncancerous tissue were frozen at –80°C. DNA was extracted using the Wizard SV Genomic DNA Purification System (Promega Corporation, Madison, WI). Loss of heterozygosity (LOH) analysis at the Ptc1 locus was done by amplification and sequencing of exon 23—from genomic DNA and BCC samples—of the murine Ptc1 gene, which holds a T/C polymorphic site at position 4016 (C, CD1 derived; T, 129Sv derived). The primers used were primer pair 8F/9R (24). Sequencing reactions were done by means of dye terminator chemistry using a Big Dye Terminator Version 3.1 Sequencing Cycle kit and a 3730 DNA Analyzer (Applied Biosystems, Foster City, CA). Loss of the CD1-derived wild-type allele in neoplastic compared with nonneoplastic tissue was considered LOH.
RNA extraction and reverse transcription-PCR. Total RNA was isolated from macroscopic BCC-like tumors using NucleoSpin Extract (Macherey-Nagel, Duren, Germany) and stored at –80°C until further processing. Total RNA (2 µg) was reverse transcribed using RETROscript (Ambion, Inc., Austin, TX) according to the instructions of the manufacturer. The primer pairs used were sense 5'-AGACTGCCGCTGGGATGGT-3' and anti–sense 5'-CGATGCCGCTTGGTCACG-3' corresponding to the Mus musculus Gli1 cDNA, yielding a 489-bp PCR product; sense 5'-CAGAACGCGGGCTTTGGTC-3' and anti-sense 5'-GGAATTCCGGGGTGTGGTG-3' corresponding to the M. musculus Gli2 cDNA, yielding a 540-bp product. In the same reaction, ß-actin-specific primers giving a 500-bp product were used as internal control. Scanning of the ethidium bromide gels was done with Electrophoresis Documentation and Analysis System (EDAS 290, Eastman Kodak Co., Rochester, NY); data were analyzed for mass and molecular weight using the 1D automatic lane and band finder (Image Analysis software, Eastman Kodak). For each sample, three different PCR amplifications were done and means ± SE were calculated.
Statistics. Analyses were done using GraphPad Prism version 4.02 for Windows (GraphPad Software, San Diego, CA). We used the Mann-Whitney test for comparison of tumor multiplicity and Fisher's exact test for analysis of tumor incidence. All P values are for two-sided tests; P < 0.05 was considered statistically significant.
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Results |
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10% of neonatal hair follicles had reached their maximal length, with location of the dermal papillae close to the s.c. muscle layer, whereas the remaining follicles showed elongation of the inner root sheath halfway up through the follicle and enlarged bulbs (Fig. 1B). These features were indicative of an early anagen phase (18). At P35 (A2), the skin showed the typical architecture of a mature anagen phase, characterized by maximal extension of hair follicles, and location of the dermal papillae in the deep subcutis (Fig. 1C). In marked contrast, in the skin of P60 mice (T2), the follicles extended no deeper than the upper dermis and resided entirely in the dermis as small fingers of quiescent epithelial cells (Fig. 1D). Hair cycling resulted in remarkable changes in mouse skin thickness; in fact, A2 skin (946 µm) and A1 skin (547 µm) were 3.5-fold and 2-fold thicker than T2 skin (275 µm), respectively. Thus, the anagen skin of Ptc1neo67/+ mice represents quite a different target for radiation compared with telogen skin.
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Two different BCC subtypes in irradiated Ptc1neo67/+ mice. All grossly visible BCC-like tumors were macroscopically similar, appearing as well-circumscribed superficial dermal nodules (Fig. 2F). Histopathologically, some of the tumors were characterized by the presence of irregular basaloid cell nests with typical peripheral cell palisading and lack of characteristics of the hair follicle lineage, resembling human BCC (Fig. 3A ; ref. 10). Interestingly, in numerous other cases, tumors had different histopathologic features, being composed by well-demarcated tumor nodules consisting of various-sized lobular nests of epithelial cells with abundant homogeneous or ground-glass pale cytoplasm, embedded in relatively scarce tumor stroma (Fig. 3B). Areas composed of elongated or spindle-shaped cells were present in some of the tumors (Fig. 3B, inset). Nuclei were basophilic and nuclear pleomorphism was mild, but mitotic figures were numerous (Fig. 3C). Prominent cleaving between tumor stroma and surrounding connective tissue was often observed (Fig. 3B and D); similar retraction spaces are frequently observed in human BCC nodules (5). In contrast with the typical BCC-like tumors, peripheral palisading of the cells was lacking in the tumor nests and blood vessels were scarce in the stroma surrounding the lobules (Fig. 3B). The peculiar morphologic characteristics of this previously undetected BCC variant were suggestive of myoepithelial differentiation, a feature also observed in human BCC (25, 26). The cellular morphology in this murine basal tumor was strikingly similar to that in the outer root sheath, the outermost compartment of the anagen hair follicle (Fig. 3D). Interestingly, we occasionally observed rare initial phases of this histologic type arising in irradiated skin of Ptc1neo67/+ mice in the approximate location where outer root sheath development occurs (Fig. 3E) and, in some cases, a direct connection with a developing follicle could be detected (Fig. 3F).
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The two histologic subtypes seemed to have a nonrandom distribution in the groups. In particular, most of the tumors (12 of 19) from A1-irradiated mice were of the newly detected SMA-expressing subtype. All other tumors from this and other groups lacked SMA expression and were classified as typical BCC-like tumors based on their strong resemblance to infiltrative human BCC and on SMA-negativity (10). In addition, SMA-expressing basal tumors appeared as an earlier wave compared with typical BCC-like tumors. They had a mean latency of 26 weeks, thus representing the vast majority (10 of 12) of the total tumors observed up to 50 weeks postirradiation (Fig. 3K). Conventional BCC-like tumors had a later onset, with a mean latency of 51 weeks, and represented the prevalent subtype (6 of 7) beginning from 50 weeks postirradiation (Fig. 3K). Collectively, the results of morphologic and histochemical analysis and the different tumor behavior support classification in two distinct BCC subtypes.
Cell lineage selection in different BCC subtypes. Immunohistochemical profiles of the two BCC subtypes and of normal-appearing skin of Ptc1neo67/+ mice help to elucidate cell lineage relationships. Consistent with the fact that actin is only sparsely found in normal epithelial cells (27), analysis of anti-SMA reactivity in normal skin showed lack of SMA expression in normal keratinocytes of the basal layer whereas SMA was detected in the arrector pili muscles and pericytes around blood vessels (Fig. 4A ). Surprisingly, both the outer root sheath of anagen hair follicles (Fig. 4B) and the follicular bulge region (Fig. 4A and D) also consistently showed strong SMA expression, suggesting a relation between SMA-expressing tumors and cells of the outer root sheath and/or the bulge. SMA expression pattern in outer root sheath cells was comparable with that of CK14 (Fig. 4B and E), and SMA intracytoplasmic reactivity was clearly in association with outer root sheath keratinocytes specifically and not, as previously suggested (28), with the fibroblasts of the dermal sheath surrounding the outer root sheath, which were uniformly SMA negative (Fig. 4B). SMA expression in the outer root sheath and bulge was not in correlation with the Ptc1 genotype, as similar SMA-positive staining of these regions was observed in wild-type CD1 mice (not shown).
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Allelic imbalance analysis at the Ptc1 locus in basal tumors. Loss of the wild-type Ptc1 allele was examined in both BCC histologic variants. Sequence analysis of tumor DNA to screen a T/C polymorphism in position 4016 of the Ptc1 gene was carried out to assess the presence of the nucleotide sequence of the targeted Ptc1 allele, derived from 129Sv embryonic stem cells, and of the wild-type allele derived from CD1 mice. We consistently detected loss of wild-type Ptc1 and retention of the mutant allele in typical BCC-like tumors (3 of 3 from different groups) and in SMA-expressing BCCs (5 of 5 from A1-irradiated mice) but not in normal control tissue (Fig. 5A and B ).
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Discussion |
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Because cellular radiosensitivity is increased in proliferating cells (20, 22), we suggest that such higher BCC susceptibility is due to the transient proliferation of follicular stem cells during anagen and to the drastic increase in number of rapidly cycling committed progenitors, or transit-amplifying cells, in individual hair follicles at this stage (32). This hypothesis is consistent with our finding that in A2-irradiated mice, the macroscopic tumor yield was only modestly increased over the T2 group, and enhancement of microscopic tumors, although significant, was less pronounced than in the A1 group. In fact, in A1 skin, only a minority of neonatal hair follicles has reached maximal length, compared with A2 skin characterized by maximal extension of hair follicles and postmitotic outer root sheath (Fig. 1C). Although hair cycle–dependent susceptibility to squamous cell papillomas developing after one-stage or two-stage chemical carcinogenesis has been reported (33), our findings show for the first time that X-ray irradiation enhances BCC development in a hair cycle–dependent manner and underscore the role of bulge stem cells in formation of basal tumors.
Development of different BCC subtypes in irradiated Ptc1neo67/+ mice. An unexpected and interesting outcome of our experiments was the qualitative difference in tumorigenicity depending on hair cycle phase at irradiation. In fact, we observed development of two different histologic BCC subtypes, both closely mimicking human tumor counterparts as regards morphologic and histochemical features, local invasiveness, and lack of metastatic spreading. BCC-like tumors resembling human BCC, previously induced in this mouse model after exposure to UV or ionizing radiation (9, 10), showed delayed onset, slow growth rate, and development through a multistep process in which nodular and infiltrative BCC-like tumors represent different stages of tumor progression. In addition, they were uniformly CK14 positive and SMA negative.
Here, for the first time, we show a high incidence of BCC-like tumors showing histologic features reminiscent of myoepithelial differentiation, occasionally described in sporadic human BCC, and SMA immunoreactivity. Interestingly, SMA-expressing BCC-like tumors had a peculiar biological behavior, characterized by fast growth rate and lack of identifiable precursor lesions, as shown by the virtually complete absence of early SMA-expressing lesions in routine microscopic observation of irradiated skin. They were also strictly hair cycle dependent, occurring only in mice irradiated in early anagen (A1), and had a mean latency considerably shorter compared with the conventional BCC subtype (26 versus 51 weeks).
Origin of BCC subtypes. An important issue is the differentiation of the starting point of tumorigenesis. To this end, morphologic and immunohistochemical analysis of tumors and of normal skin from experimental mice has provided several important cues about the nature of cellular targets for the different tumors arising in irradiated Ptc1neo67/+ mice.
Our data suggest a critical role for the interfollicular epidermis in formation of conventional BCC-like tumors. The close anatomic connection of interfollicular epidermis with nodular BCCs—precursors of conventional BCC-like tumors—suggests that these tumors derive from interfollicular epidermis as epidermal downgrowths into the underlying dermis (Fig. 4H). Similarly, previous studies have shown that the epithelium of follicular hamartomas, also arising in association with deregulated Shh signaling, is connected to epidermis rather than hair follicles both in humans and in mice (29).
We show here that CK15-expressing epidermal cells are present in adult epidermis of Ptc1neo67/+ mice, consistent with recent results showing that label-retaining cells are found within the basal layer of the epidermis in addition to the hair follicle bulge (34). Because CK15 is considered a biochemical marker of epidermal stem cells (15), interfollicular CK15-expressing cells may represent the target for nodular BCC and its progressed stage, the macroscopic typical BCC. Although CK15 is reported as a good intracellular marker for both the stem and transit-amplifying population in human skin (35), it cannot distinguish between keratinocyte stem cells and the transit-amplifying progeny of bulge cells in CK15-positive epidermal cells. Because epidermis growth is continuous rather than cyclical, the strong hair cycle–dependent enhancement of nodular BCC—the early-stage lesion for the macroscopic typical BCC-like tumor—suggests a contribution of follicular bulge stem cells to this tumor subtype. This is consistent with earlier results showing that bulge epidermal stem cells are not only responsible for forming the lower hair follicle but may be also a major source of progenitor cells that give rise to the interfollicular, cornified epithelium (11).
A distinct cell-target population can be postulated for SMA-expressing, nonconventional BCC-like tumors, which could explain their distinctive biological features. Based on morphologic and immunohistochemical evidence, we propose that this population is represented by the outer root sheath progenitors, which undergo maximal transient proliferation during anagen to generate a critical part of the new hair follicle structure, and have been shown to express potential markers of hair follicle stem cells (36).
Thus, our findings are suggestive of a different cellular origin of the two BCC subtypes. However, at this stage, we cannot exclude that both types of BCCs arise from the same lineage (i.e., from bulge stem cell progeny) and what distinguishes them is that they arise from cells in different states of differentiation.
The biological significance of the presence of SMA in outer root sheath cells remains unknown. Because actin is the predominant component in contractile microfilaments, which are responsible for cell motility and transport, SMA may play a role in cell migration and/or morphologic changes in the lower portion of the follicle during hair cycle (28). On the other hand, because the follicular bulge region also showed SMA immunoreactivity (Fig. 4A), it is possible that SMA-expressing cells in the outer root sheath (and possibly in outer root sheath-related tumors) derive from SMA-expressing putative stem cells of the bulge area. As SMA expression was reported in smooth muscle progenitor cells (37), SMA expression in the bulge might be analogous to the reported expression of nestin, the neural stem cell marker, in bulge stem cells (36). Indeed, nestin-expressing cells isolated from the mouse hair-follicle bulge area were shown to be able to differentiate in vitro into many cell lineages, including smooth muscle cells (38). Our finding that cells of the bulge area express SMA in vivo strongly supports the in vivo multipotency of hair follicle stem cells.
Analysis of Ptc1 allelic status. Analogously to human BCCs, LOH at the Ptc1 locus is implicated in BCC tumorigenesis in Ptc1neo67/+ mice and has been reported in previous experiments with UV (9) or ionizing radiation (10, 39). To determine whether the different histologic features of BCC subtypes were associated with altered allelic patterns at the Ptc1 locus, we examined Ptc1 allelic imbalance in DNAs from both BCC variants. We show that all tumors analyzed are characterized by loss of the remaining normal Ptc1 allele, confirming that complete loss of Ptc1 function is a key event in BCC tumorigenesis regardless of the differentiation route or cellular target involved in tumor formation.
Expression of Gli1 and Gli2 in BCC subtypes. Several mouse models support the concept that aberrant Shh pathway activation is sufficient to drive development of BCC-like tumors (5, 6, 9, 40, 41), and recent results indicate that different levels of Shh signaling activity results in formation of different tumor types in skin (29). Uncontrolled Shh signaling associated with BCC development is mediated primarily through the Gli family of transcription factors and BCC-like tumors are induced by overexpression of Gli1 and Gli2 in mouse epidermis (5, 6). Particularly, Gli1 overexpression results in development of multiple types of skin tumors of basal cell origin, only a minority of which are BCCs (5). In contrast, overexpression of Gli2 causes spontaneous development of multiple BCCs (6, 41). This is interesting in light of our results of expression of Gli1 and Gli2 in radiation-induced typical BCC-like tumors and SMA-expressing variants. Whereas Gli1 was similarly expressed in both tumor subtypes, Gli2 mRNA expression was significantly higher in typical BCC-like tumors. Whether this reflects a different expression level of the two Shh-signaling mediators in the target cell populations involved in tumor formation or, rather, a dependence of tumor phenotype on the level of Shh activity regardless of the tumor progenitor cells remains to be established. However, based on our results and on recent findings showing that Gli2, but not Gli1 mRNA, is expressed in normal interfollicular epidermis (42), we favor the hypothesis that differential Gli2 expression in BCC subtypes developing in irradiated Ptc1neo67/+ mice reflects the expansion of different cellular targets and/or progenitor cells at different stages of hair follicle maturation.
Conclusions. The studies outlined here support the concept that BCC tumorigenesis can be strongly affected by the hair growth cycle and that the tumor yield, histologic type, and tumor behavior are dependent on the follicular phase during which tumor initiation by radiation occurs. By showing that the time periods of susceptibility to different BCC subtypes coincide with the transient proliferation of stem cells/committed progenitors, and by defining histochemical relationships between putative target cells and BCC variants, our results contribute to clarify the lineage selection observed in tumors. Finally, by delineating a new smooth muscle–related histochemical profile for bulge and outer root sheath cells, we extend previous findings of neural stem cell marker expression in bulge/outer root sheath progenitors (36) and strengthen the hypothesis that these cells represent a multipotent adult stem-cell compartment that can easily generate cells of more than one embryonic lineage.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Orsio Allegrucci for assistance in handling and treating the mice.
Received 10/12/05. Revised 3/31/06. Accepted 4/26/06.
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References |
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-smooth muscle actin in basal cell epithelioma and its surrounding stroma: with special reference to proliferating cell nuclear antigen expression and adenoid differentiation. Br J Dermatol 1994;130:189–94.[Medline]This article has been cited by other articles:
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  M. Tanori, M. Mancuso, E. Pasquali, S. Leonardi, S. Rebessi, V. Di Majo, M.-N. Guilly, F. Giangaspero, V. Covelli, S. Pazzaglia, et al. PARP-1 cooperates with Ptc1 to suppress medulloblastoma and basal cell carcinoma Carcinogenesis, October 1, 2008; 29(10): 1911 - 1919. [Abstract] [Full Text] [PDF]
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 K. Tanese, M. Fukuma, T. Yamada, T. Mori, T. Yoshikawa, W. Watanabe, A. Ishiko, M. Amagai, T. Nishikawa, and M. Sakamoto G-Protein-Coupled Receptor GPR49 is Up-regulated in Basal Cell Carcinoma and Promotes Cell Proliferation and Tumor Formation Am. J. Pathol., September 1, 2008; 173(3): 835 - 843. [Abstract] [Full Text] [PDF]
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X174 DNA/BsuRI (HaeIII) Marker, 9, Fermentas International, Inc., Burlington, Ontario, Canada]. E, normalized expression levels of Gli1 and Gli2 in typical BCC-like tumors (SMA–) and SMA-expressing variants (SMA+). Three independent PCR runs were carried out for each tumor and ß-actin was used as a reference standard for all analyses to control for the amount of sample material. Data represent the ratios of final averages for BCC-like tumors of each subtype (n = 5). Bars, SD.